Alzheimer's Disease (AD) is the most common form of dementia that affects 5.3 million Americans. In a small percentage (>1%) of cases AD is inherited as an autosomal dominant trait (Familial AD), however the majority of cases are sporadic. A key neuropathological event in AD is the cerebral accumulation of A, a ~4kDa peptide derived by serial proteolysis of the amyloid precursor protein (APP) by - and ?-secretase. Beta-site APP-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as -secretase. Several studies have shown that BACE1 protein levels and-secretase activity are increased in AD brains. Thus, BACE1 elevation may be the first step in increasing A and triggering AD pathology, at least in the sporadic cases. Our studies have elucidated a novel post-translational mechanism of regulation of BACE1 mediated by the BACE1-interacting molecule, GGA3 (Golgi-localized ?-ear-containing ARF binding protein 3). We have determined that GGA3 depletion stabilizes BACE1 and increases -secretase activity. We also found that levels of GGA3 are decreased in post-mortem AD brains and are inversely correlated with BACE1 levels. We have shown that BACE1 is degraded via the lysosomal pathway and demonstrated that GGA3 regulates the delivery of BACE1 to the lysosomes. The BACE1-C-terminal fragment (CTF) contains a specific di-leucine (DXXLL) sorting signal that has been shown to bind the VHS domain of the three members of the GGA family of proteins, GGA1, 2, and 3. We have found that, unexpectedly, direct binding of GGA3 VHS domain to the BACE1 di-leucine motif is not necessary for this regulation. Instead, GGA3 interaction with ubiquitin is essential for regulating BACE1 levels. Accordingly, we have found that BACE1 is mainly mono- and K63-linked polyubiquitinated at lysine 501. The central hypothesis of this proposal is that the impairment of BACE1 degradation is the underlying mechanism of BACE1 elevation in the brains of subjects affected by AD. The overarching goal of this proposal is to determine the extent to which GGA- and ubiquitin-mediated regulation of BACE1 represent a potential target for the treatment of AD. Thus, we propose to specifically address the following aims: 1) To determine the extent to which BACE1 ubiquitination regulates BACE1 trafficking, activity and degradation via the proteasomal or lysosomal pathway; 2) To determine the extent to which over-expression of GGA3 reduces levels of BACE1 and A in a ubiquitin-dependent fashion in vivo; 3) To determine the extent to which GGA1, another member of the GGA family of proteins, regulates levels of BACE1 and A independently and in association with GGA3 in vitro and in vivo.